Frank J. Burczynski

Enterohepatic circulation: Physiological, pharmacokinetic and clinical implications

Enterohepatic recycling occurs by biliary excretion and intestinal reabsorption of a solute, sometimes with hepatic conjugation and intestinal deconjugation. Cycling is often associated with multiple peaks and a longer apparent half-life in a plasma concentration-time profile. Factors affecting biliary excretion include drug characteristics (chemical structure, polarity and molecular size), transport across sinusoidal plasma membrane and canniculae membranes, biotransformation and possible reabsorption from intrahepatic bile ductules. Intestinal reabsorption to complete the enterohepatic cycle may depend on hydrolysis of a drug conjugate by gut bacteria. Bioavailability is also affected by the extent of intestinal absorption, gut-wall P-glycoprotein efflux and gut-wall metabolism.Recently, there has been a considerable increase in our understanding of the role of transporters, of gene expression of intestinal and hepatic enzymes, and of hepatic zonation. Drugs, disease and genetics may result in induced or inhibited activity of transporters and metabolising enzymes. Reduced expression of one transporter, for example hepatic canalicular multidrug resistance-associated protein (MRP) 2, is often associated with enhanced expression of others, for example the usually quiescent basolateral efflux MRP3, to limit hepatic toxicity. In addition, physiologically relevant pharmacokinetic models, which describe enterohepatic recirculation in terms of its determinants (such as sporadic gall bladder emptying), have been developed.In general, enterohepatic recirculation may prolong the pharmacological effect of certain drugs and drug metabolites. Of particular importance is the potential amplifying effect of enterohepatic variability in defining differences in the bioavailability, apparent volume of distribution and clearance of a given compound. Genetic abnormalities, disease states, orally administered adsorbents and certain coadministered drugs all affect enterohepatic recycling.

Antioxidative function of L‐FABP in L‐FABP stably transfected Chang liver cells

Liver fatty acid binding protein (L‐FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L‐FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L‐FABP mRNA level. Full‐length L‐FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA‐FABP). Chang cells were stably transfected with pc‐DNA‐FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 μmol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L‐FABP was documented by regular reverse transcription polymerase chain reaction (RT‐PCR), real‐time RT‐PCR, and Western blot. The pcDNA‐FABP–transfected cells expressed full‐length L‐FABP mRNA, which was absent from vector‐transfected control cells. Western blot showed expression of 14‐kd L‐FABP protein in pcDNA‐FABP–transfected cells, but not in vector‐transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L‐FABP expression. Lower LDH release was observed in the higher L‐FABP–expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L‐FABP gene. The L‐FABP–expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L‐FABP has a significant role in oxidative stress. (HEPATOLOGY 2005;42:871–879.)

Antibiotic-Heparin Lock: In Vitro Antibiotic Stability Combined with Heparin in a Central Venous Catheter

Long‐term hemodialysis frequently requires vascular access through central venous catheters (CVCs). Infection related to these catheters is a significant complication. The use of an antibiotic‐heparin lock could decrease the risks associated with infected permanent catheters. As an initial step in developing an antibiotic‐heparin lock, we investigated the in vitro stability of antibiotic‐heparin combinations in CVCs. Initially, cefazolin, vancomycin, ceftazidime, ciprofloxacin 10 mg/ml each, and gentamicin 5 mg/ml were incubated separately in glass test tubes in the dark at 37°C for 72 hours. Samples were analyzed spectrophotometrically for stability at 24‐hour intervals. The procedure was repeated with the addition of heparin (final concentration 5000 U/ml in glass test tubes), and the combination was also examined in CVCs. High‐performance liquid chromatography analysis was conducted on the antibiotic‐heparin combinations at 72 hours to confirm the spectrophotometric results. Ciprofloxacin produced an immediate precipitate with the addition of heparin and was not analyzed further. Absorbance values decreased for all antibiotics, with the greatest decreases at 72 hours for cefazolin (27.4%), vancomycin (29.7%), ceftazidime (40.2%), and gentamicin (8%) when combined with heparin. These decreases were postulated to be secondary to adsorption of the antibiotics to the luminal surface of the catheters because submitting the catheters to ultrasound with 1% sodium bicarbonate and analyzing the resulting solution for absorbance revealed that some of the drug was recovered. Although free antibiotic in CVC solution was reduced, the concentration should be sufficient (approximately 5 mg/ml) to decrease the frequency of infections associated with CVCs. We conclude that the concentrations of vancomycin, ceftazidime, cefazolin, or gentamicin used in our study should be sufficient for an antibiotic‐heparin lock.

Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity.

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of ∼80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or α-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABPs antioxidant activity is through inactivation of the free radicals by L-FABPs methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.

Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay

The oxidation of 2′,7′-dichlorodihydrofluorescein (2′,7′-dichlorofluorescin, DCFH) to a fluorescent product, 2′,7′-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates.

Development of a Nitric Oxide-Releasing Analogue of the Muscle Relaxant Guaifenesin for Skeletal Muscle Satellite Cell Myogenesis

Nitric oxide (NO) mediates activation of satellite precursor cells to enter the cell cycle. This provides new precursor cells for skeletal muscle growth and muscle repair from injury or disease. Targeting a new drug that specifically delivers NO to muscle has the potential to promote normal function and treat neuromuscular disease, and would also help to avoid side effects of NO from other treatment modalities. In this research, we examined the effectiveness of the NO donor, iosorbide dinitrate (ISDN), and a muscle relaxant, methocarbamol, in promoting satellite cell activation assayed by muscle cell DNA synthesis in normal adult mice. The work led to the development of guaifenesin dinitrate (GDN) as a new NO donor for delivering nitric oxide to muscle. The results revealed that there was a strong increase in muscle satellite cell activation and proliferation, demonstrated by a significant 38% rise in DNA synthesis after a single transdermal treatment with the new compound for 24 h. Western blot and immunohistochemistry analyses showed that the markers of satellite cell myogenesis, expression of myf5, myogenin, and follistatin, were increased after 24 h oral administration of the compound in adult mice. This research extends our understanding of the outcomes of NO-based treatments aimed at promoting muscle regeneration in normal tissue. The potential use of such treatment for conditions such as muscle atrophy in disuse and aging, and for the promotion of muscle tissue repair as required after injury or in neuromuscular diseases such as muscular dystrophy, is highlighted.

In vitro evaluation of concurrent use of commercially available insect repellent and sunscreen preparations

Background  Insect repellents and sunscreens are over‐the‐counter products extensively used by the general public. Concurrent application of these products has become widespread in many regions across North America, because of concerns about West Nile virus and skin cancers.

Infection of myocytes with chlamydiae.

Chlamydial infection has been associated with myocarditis in animals and humans. However, the mechanism resulting in myocarditis following infection is not known. Here, evidence is presented that both Chlamydia trachomatis and Chlamydia pneumoniae can infect and replicate in myocytes isolated from neonate rats. The infected myocytes contained chlamydial inclusions, indicative of chlamydial growth, and infectious particles were recovered from the infected myocytes. It was also found that chlamydial infection at a late stage induced significant damage to the infected myocytes, as evidenced by an increased lactate dehydrogenase release, reactive oxygen species production and a reduced ATP level. However, no nuclear apoptosis was detected in the infected myocytes. Collectively, these observations have demonstrated that Chlamydia spp. are able to both infect and damage myocytes, suggesting a potential role of chlamydial infection in myocarditis.

The Role of Extracellular Binding Proteins in the Cellular Uptake of Drugs: Impact on Quantitative In Vitro-to-In Vivo Extrapolations of Toxicity and Efficacy in Physiologically Based Pharmacokinetic-Pharmacodynamic Research.

A critical component in the development of physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) models for estimating target organ dosimetry in pharmacology and toxicology studies is the understanding of the uptake kinetics and accumulation of drugs and chemicals at the cellular level. Therefore, predicting free drug concentrations in intracellular fluid will contribute to our understanding of concentrations at the site of action in cells in PBPK/PD research. Some investigators believe that uptake of drugs in cells is solely driven by the unbound fraction; conversely, others argue that the protein-bound fraction contributes a significant portion of the total amount delivered to cells. Accordingly, the current literature suggests the existence of a so-called albumin-mediated uptake mechanism(s) for the protein-bound fraction (i.e., extracellular protein-facilitated uptake mechanisms) at least in hepatocytes and cardiac myocytes; however, such mechanism(s) and cells from other organs deserve further exploration. Therefore, the main objective of this present study was to discuss further the implication of potential protein-facilitated uptake mechanism(s) on drug distribution in cells under in vivo conditions. The interplay between the protein-facilitated uptake mechanism(s) and the effects of a pH gradient, metabolism, transport, and permeation limitation potentially occurring in cells was also discussed, as this should violate the basic assumption on similar free drug concentration in cells and plasma. This was made because the published equations used to calculate drug concentrations in cells in a PBPK/PD model did not consider potential protein-facilitated uptake mechanism(s). Consequently, we corrected some published equations for calculating the free drug concentrations in cells compared with plasma in PBPK/PD modeling studies, and we proposed a refined strategy for potentially performing more accurate quantitative in vitro-to-in vivo extrapolations (IVIVEs) of toxicity (efficacy) at the cellular level from data generated in cell assays. Overall, this present study may help to optimize the human dose prediction in preclinical and clinical studies, while prescribing drugs with narrow therapeutic windows that are highly bound to extracellular proteins and/or highly ionized at the physiological pH. This may facilitate building a more accurate safety (efficacy) profile for such drugs.

Recent insights into the biological functions of liver fatty acid binding protein 1.

Over four decades have passed since liver fatty acid binding protein (FABP)1 was first isolated. There are few protein families for which most of the complete tertiary structures, binding properties, and tissue occurrences are described in such detail and yet new functions are being uncovered for this protein. FABP1 is known to be critical for fatty acid uptake and intracellular transport and also has an important role in regulating lipid metabolism and cellular signaling pathways. FABP1 is an important endogenous cytoprotectant, minimizing hepatocyte oxidative damage and interfering with ischemia-reperfusion and other hepatic injuries. The protein may be targeted for metabolic activation through the cross-talk among many transcriptional factors and their activating ligands. Deficiency or malfunction of FABP1 has been reported in several diseases. FABP1 also influences cell proliferation during liver regeneration and may be considered as a prognostic factor for hepatic surgery. FABP1 binds and modulates the action of many molecules such as fatty acids, heme, and other metalloporphyrins. The ability to bind heme is another cytoprotective property and one that deserves closer investigation. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation and the adverse effects of starvation (energy deficiency).